1. Field of the Invention
The present invention relates to a maltopentaose (G5) producing amylases and derivatives thereof.
2. The Prior Art
Apart from glucose (glucoamylases) and maltose (.beta.-amylases), only very few maltooligosaccharides can be obtained directly in sufficient purity by hydrolysis of starch using amylases. On hydrolysis of starch, as a rule, .alpha.-amylases produce mixtures of glucose and lower molecular weight maltooligosaccharides (G2-G9). Purification of individual components from such mixtures is elaborate and costly. However, individual .alpha.-amylases have sufficiently high product specificity to enable the industrial production of defined oligosaccharides. To date, 3 G5 forming amylases have been disclosed:
(a) Bacillus licheniformis: U.S. Pat. No. 4,039,383, issued Aug. 2, 1977; Arch. Biochem. Biophys., 155, 290-298, (1973)
The enzyme from the thermophilic organism Bacillus licheniformis has a temperature optimum of 70.degree. C. and is active in a wide pH range of pH 4.0-10.0. Its molecular weight (MW) is 22.5 kDa. The initial products of amylose hydrolysis are long chain maltooligosaccharides (G5-Gn) which are, however, further degraded as the reaction progresses to the main product G5 and, in relatively large amounts, also to G1-G4. U.S. Pat. No. 4,039,383 of Aug. 2, 1977, describes a process for the hydrolysis and solubilization of amylose (a substrate of low solubility in water). The dissolved amylose is then used as substrate for the purified amylase to produce G5. Because of the many byproducts, the mixture of products after the enzyme reaction must be purified by chromatography.
(b) Bacillus cereus NY-14: Japanese Patent No. 158,099, of Sep. 13, 1982, Which Corresponds to U.S. Pat. No. 4,591,561, of May 27, 1986; Japanese patent No. 142,330, of Aug. 3, 1983; Agric. Biol. Chem., 49 (12), 3369-3376, (1985) (ABC)
The indicated citation (ABC) describes the purification and characterization of a 55 kDa amylase from Bacillus cereus NY-14, which shows maximal activity at pH 6.0 and 55.degree. C. The enzyme cleaves starch initially into the maltooligosaccharides G3-G8. The long chain sugars are then subsequently degraded to G1-G5. Japanese Patent No. 158,099, of Sep. 13, 1982, which corresponds to U.S. Pat. No. 4,591,561, of May 27, 1986, describes the production of G5 by culturing a Bacillus strain (NY-14 in this case) in a medium which contains a substrate (starch, amylose, etc.) which can be cleaved into maltooligosaccharides by enzymes which are produced by the organism used. In this process, defined oligosaccharides are obtained by filtration of the culture broth and subsequent chromatography. Japanese Patent No. 142,330 of Aug. 3, 1983, describes the G5-specific enzyme from Bacillus cereus NY-14. There is a contradiction in the description of the enzyme to the description in ABC, because the stated MW of the enzyme is 90 kDa in the patent, but is 55 kDa in the publication.
(c) Pseudomonas sp. KO 8940: Japanese Patent No. 44,069, of Mar. 9, 1984; Japanese Patent No. 44,070 of Mar. 9, 1984; Japanese patent No. 253786-87 (Div ex 44069-84); Appl. Microbiol. Biotechnol., 25, 137-142, (1986); Agric. Biol. Chem., 54 (1), 147-156. (1990)
The authors of the Appl. Microbiol. Biotechnol. describe primarily the Pseudomonas isolate KO 8940 and the conditions necessary for production of a G5-amylase. The most recent publication (Agric. Biol. Chem. 54 (1), 147-156 (1990)) describes the purification and biochemical characterization of probably this G5-amylase. The amylase from the Pseudomonas isolate KO 8940 is, however, not expressly mentioned. The purified enzyme has a high initial G5-forming activity. Shorter hydrolysis products occur only after prolonged incubation times. Japanese Patent No. 253786-87 describes the enzyme from Pseudomonas KO 8940 and its use for producing G5. According to this Japanese patent, the amylase has an optimum temperature of 45.degree. C. to 55.degree. C. and an optimum pH of pH 6.0-7.0. Its MW is 72.5 kDa.
Japanese Patent No. 44,070 of Mar. 9, 1984, discloses the amylase producer Pseudomonas KO 8940.
To obtain maltopentaose using the known enzymes, either elaborately purified enzymes are used, or the maltopentaose is elaborately purified from the culture substrate.